Failure of the lung endothelial barrier leads to pulmonary edema and acute lung injury (All). Mechanical ventilation contributes to all mortality by subjecting the lung to excessive ventilatory stretch (VS), causing ventilator induced lung injury (VILI). In animal models of VILI, pulmonary endothelial barrier dysfunction is associated with increased endogenous nitric oxide (NO) production. NO activates endothelial soluble guanyl cyclase (sGC), producing cyclic guanosine monophosphate (cGMP). The effect of endothelial cGMP on VS-induced endothelial barrier function is unclear. Our preliminary experiments in a mouse model of VILI suggest that VS activates endothelial sGC to increase cGMP. After VS onset, sGC inhibition or stimulation attenuates or exacerbates barrier dysfunction, respectively, suggesting an injurious role for cGMP. In contrast, sGC activation before VS is barrier protective. Increasing endothelial cGMP production by non- sGC mechanisms (particulate guanylyl cyclase, pGC) is protective of the endothelial barrier, regardless of timing with the onset of VS. These and other data suggest an intracellular compartmentalization of sGC- mediated cGMP synthesis that is altered by VS to change the effect of cGMP signaling on barrier function, possibly by translocation of sGC from the cytosol to a caveolar membrane domain. The goal of this application is to determine the effect of cGMP on VILI with a specific focus on cGMP compartmentalization. In Aim 1, the effect of sGC-produced cGMP on endothelial barrier function will be further characterized in an isolated perfused mouse lung preparation. The dose-response and specificity of sGC activation on cGMP production and endothelial barrier dysfunction will be determined. In addition, the timing of the VS-induced switch from cGMP protection to harm will be determined. In Aim 2, the effect of lung VS on the subcellular compartmentalization of endothelial sGC will be determined in both intact mouse lungs and cyclically- stretched endothelial cells by confocal microscopy and subcellular fractionation. The barrier effect of sGC- caveolae interaction will be determined in isolated, perfused caveolin-1 -/-mouse lungs. VS-induced interactions of sGC with phosphodiesterase 2, a key effector of cGMP-mediated endothelial barrier dysfunction, will be explored using specific inhibitors, immunoprecipitation, and cAMP measuremnts. Relevance: Acute lung injury from infection or trauma causes lung failure because the endothelial cells lining blood vessels allow fluid to leak into the air spaces. Mechanical ventilatory support is vital, but over- distension of lung tissue can worsen injury through changes in endothelial molecules such as cGMP. Understanding how cGMP worsens endothelial leakiness could lead to better therapies of acute lung injury. [unreadable] [unreadable] [unreadable] [unreadable]